IACUC GUIDELINES FOR THE USE OF FRUEND'S COMPLETE
ADJUVANT IN RODENTS AND RABBITS
The purpose of performing immunization protocols is to generate and study immune
responses. Pain and distress to the animal must be addressed as many of the standard
practices used in immunological research have the potential to produce pain and distress if
they are not done correctly. The following guidelines, approved by the School of Medicine
Animal Care and Use Committee, are intended to inform investigators of humane methods
for conducting these protocols.
Freund's complete adjuvant (FCA) is a water in oil emulsion containing killed, dried
Mycobacterium butyricum which is used to enhance antigenicity and stimulate an immune
response greater than antigen alone both in duration and magnitude. The disadvantage to
this agent is that the adjuvant can induce aggressive and persistent granulomatous lesions
when used intradermally or subcutaneously. Intramuscular injection may cause permanent
lameness. Intravenous injection may damage the lungs. Intraperitoneal injection, used in
mice and rats, causes granulomatous peritonitis, but may not necessarily cause chronic
distress. Because of the detrimental effects, the investigator should consider other available
adjuvants. Since the USDA has declared that the use of FCA may cause more than
momentary pain and distress, alternatives must be considered. A description of the methods
and sources used to search for alternatives to painful procedures must be provided in the
animal use protocol. This should include: databases searched or other sources consulted,
date of the search and years covered by the search, key words used to help determine that
no alternatives were available to the painful or distressful procedure (i.e. the use of FCA).
FCA is a hazardous substance. Special care must be taken to avoid parenteral exposure of
personnel involved in the preparation and administration of FCA. Accidental intradermal or
intramuscular inoculation may result in tuberculin sensitization of tuberculin negative
individuals, and moderate to severe local, regional, or systemic hypersensitivity reactions in
individuals who are sensitized to tuberculin. Inadvertent ocular exposure can lead to
blindness.
In preparing FCA, the mycobacteria should first be resuspended by vortexing or shaking.
One part or less of Freund's adjuvant to one part antigen (v/v) is recommended. Two sterile
luer-lock syringes, one with Freund's and one with the antigen preferably in sterile saline
are used. Glass syringes are preferred as the oil will react with the rubber plunger on plastic
disposable syringes. Millipore filtration of the antigen before mixing with adjuvant is
recommended.
As a general suggestion, FCA should be used for weakly immunogenic compounds or for
small amounts of immunogen. Several other synthetic, non-inflammatory adjuvants are
available which may offer advantages in some situations:
RAS (Ribi Adjuvant System, Ribi Immunochemical Research. Inc; P.O. Box 1409, Hamilton,
Montana 59840).
Hunter's TiterMax or TiterMax Gold (without silica) (CytRx, 154 Technology Parkway, N.W.,
Norcross, GA 30092)
Quil A, a saponin-type, surface-active adjuvant (Accurate Chemical Scientific Corporation,
Westbury, NY 11590)
IMMUNIZATION TECHNIQUES
FCA should be used only once, for the initial immunization in all species
The approved animal protocol form must include the site of injection, the amount of
material administered per site, the number of sites and hence the total volume injected, the
adjuvant system used, and the frequency and total number of booster injections. When
giving subcutaneous injections the use of multiple sites containing small volumes is more
beneficial from both humane and scientific perspectives as this technique distributes the
immunogen/adjuvant over a larger surface area for the immune system to process resulting
in higher titers and also reducing the incidence of severe local inflammatory lesions and
abscessation. Injection sites must be sufficiently separated to prohibit coalescing of the
inflammatory lesions. Intraperitoneal injection, which is permissible in small rodents, should
only be given as a single immunization. The volume of the peritoneal cavity limits the total
volume administered.
Intramuscular injections in the rabbit are discouraged as the inflammatory response can
cause permanent lameness and nerve damage.
Intradermal and footpad injections are generally not allowed unless scientifically justified by
the investigator and approved by the IACUC. Intradermal injections have a greater capacity
to cause local inflammatory reactions and abscessation. Footpad injection (intradermal in
theory, in practice this route is probably a mixture of intradermal and subcutaneous, and
perhaps even intravenous in unskilled hands) is discouraged due to the sensitivity of the
site especially when inflammation is present. The forepaw footpads should not be used in
mice and rats as they use the forepaws for food handling. Rabbits should not be injected in
the footpad, as they do not have true footpads.
The minimum time period between the initial and booster immunizations is usually 2-3
weeks. Booster injections should be delayed if significant inflammatory reactions are still
present from previous injections. If adjuvant-antigen mixtures have caused excessive
reactions, you should use lesser concentrations, smaller volumes per injection site, or both
for all subsequent immunizations. Veterinary consultation may be indicated. When an
animal is given a booster dose too soon, the immune response may be suppressed rather
than enhanced. Ideally, one should follow the serum antibody titer in an immunized animal
and give a booster injection of antigen only after the antibody titer has plateaued or begun
to decline. Patience should be practiced when selecting the time interval between the
primary immunization and the booster immunization.
Animals can be rested for long intervals between boosting. Even when serum antibody titers
have dropped to relatively low levels, a booster injection into an animal that had previously
established a memory response will usually reestablish a high serum antibody titer.
ANIMALS SUCH AS RABBITS AND GUINEA PIGS SHOULD BE PROVIDED WITH MILD
SEDATION AND ANALGESIA WHEN MULTIPLE INJECTIONS ARE DONE. The
administration of a 1:1 mixture of acepromazine (10mg/ml) and torbugesic (10mg/ml) at a
dosage of 0.15ml/kg (not to exceed 0.75ml) given subcutaneously or intramuscularly has
been effective for rabbits. Isoflurane anesthesia by mask is a safe method for providing
restraint in guinea pigs, and buprenorphine(.05mg/kg) SC can be given for pain.
SITE PREPARATION
Anatomic sites used for grasping, handling, or restraint should be avoided. This would
include the neck area in rabbits and rodents, and the tail base in rodents. Hair should be
clipped from intradermal and subcutaneous sites, and the sites should be aseptically
prepared with betadine or nolvasan scrub followed by alcohol. Sterile needles and syringes
should also be used.
subcutaneous intramuscular
intraperitoneal footpad
intravenous
intradermal
Rabbit
.1-.25ml per
site; 1-1.2ml
total volume;
on the back
Not allowed
Not allowed
Not allowed
Must be
justified
and
approved.
.05 ml per
site, use a
25 to 27
gauge
needle, on
the back,
no more
than 10
sites; used
for cell
mediated
immunity
Guinea
pig
Total volume
up to .4ml,
divided in 2
sites in the
dorsal neck
area
Mouse=less
than .25ml
Rat=less than
.5ml
Must be
justified
and
approved, 1
hind foot
per animal,
house on
soft
bedding
Mouse=.01.05ml
Rat=less
than .1ml
Not
recommended
and generally
not allowed
Must be
justified
and
approved;
.01-.05ml
per site
Must be
justified and
approved;
inject in
bieceps
femoris,
quadriceps or
lumbar
muscle; no
more than
.25ml; one
leg only
Rodents Mouse=.05ml Not
per site; 2-4 recommended
sites
and generally
not allowed
Rat=up to .1
ml per site;
2-4 sites
APPROXIMATE TOTAL BLOOD VOLUMES
Rabbit 56ml/kg
Mouse 78ml/kg Rat 67ml/kg
Hamster 87ml/kg
Guinea Pig 75ml/kg Dog 86ml/kg
Monkey 77ml/kg
Swine 75ml/kg
As a single blood draw less than or equal to 10 per cent of the TOTAL blood volume can be
removed. A 14-day recovery period is needed for the average animal to recover from this
blood loss.
(example: 4.5kg rabbit; total blood volume= (4.5kg) X (56ml/kg)= 252 ml total blood
volume. (0.1) X (252)=25.2ml maximum blood draw.
The use of intracardiac puncture is restricted to non-survival procedures. Anesthesia is
required for intracardiac puncture, orbital sinus puncture, and all terminal exanguination
procedures.
Guidelines for safe blood withdrawals are based on the animal's total blood volume, body
weight, and health status. These guidelines are for normal, healthy animals.
As reported in the OPRR Reports 11/17/97, "there is evidence that the mouse ascites
method of monoclonal antibody production causes discomfort, distress, or pain. Practical in
vitro methods exist which can replace the ascites method in many experimental applications
without compromising the aims of the study." An IACUC approved animal protocol that
includes mouse antibody production must scientifically justify its use, alternative or in vitro
methods must have been considered, and alternative or in vitro methods must have been
determined unsuitable.
MOUSE ASCITES PRODUCTION
1. Use healthy, viral antibody free animals for ascites production. Cell lines should be MAP
(Mouse Antibody Production) tested for pathogens before being injected into any animal.
2. Retired breeders may be superior to young animals since the abdominal distension is
better tolerated and greater volumes may be obtained.
3. Pristane is commonly used to prime the abdomen prior to implantation with a hybridoma.
Pristane is toxic at doses only slightly higher than the amount recommended for ascites
production. 0.5 ml is the historical dose, but there is good evidence that doses as low as
0.1-0.2 ml give similar results. Mice should be observed daily for signs of distress, and the
protocol must be modified accordingly.
4. Incomplete Freund's Adjuvant (dose is 0.5 ml) has also been used as a priming agent for
ascites production. Adverse side effects are usually less than with Pristane. With IFA, tumor
cell lines can be implanted as soon as 24 hours post administration.
5. The rate of ascites fluid production is extremely variable, but generally begins 1 week
following injection. Animals must be observed daily and have abdominal fluid removed at
regular intervals to prevent excessive accumulation. The ascites fluid should be collected
when the abdomen is approximately the proportion of a near-term pregnant animal. Ascites
collection should be made using a sterile 21-gauge needle (or smaller) preferably on an
anesthetized animal. The skin surface should be disinfected with 70% alcohol. 2-3 ml of
warmed saline can be given subcutaneously to the mouse at the time of paracentesis.
6. Animals in distress (cachexic, not eating, increased respiratory rate, lethargy, rough hair
coat, hunched posture, hypothermic, dehydrated, etc.) should be euthanized.
7. Determining an appropriate time for euthanasia of the ascites mouse is critical.
General guidelines recommend 2 survival taps followed by a third non-survival tap.
Approved by the IU School of Medicine IACUC June 13, 2001
REFERENCES:
ILAR JOURNAL, VOLUME 37 (3)- 1995
State University of New York; Upstate Medical University- Guidelines for the Production of
Polyclonal and Monoclonal Antibodies in Rodents and Rabbits
Cornell University- Guidelines for Production of Polyclonal Antibodies in Rodents and Rabbits
University of Florida Animal Care and Use- Guidelines for Using Freund's Adjuvant in Rabbits
Mercer University-Guidelines on Immunological Procedures; Adjuvants
NIH-Guidelines for the Use of Freund's Adjuvant in Laboratory Animals
University of Minnesota Division of Comparative Medicine
The Care and Feeding of an IACUC, p. 185-186
University of Connecticut, IACUC guidelines
OPPR Reports-November 17, 1997